DESCRIPTION: (Applicant's Abstract) In the previous stage of this project, supported by DA 08379-01 through 03 we have characterized cocaine binding sites on the dopamine transporter (DAT) in relation to sites for other uptake blockers, DA, and ions, as well as their regulation by protein kinase C and arachidonic acid. Studies included both the rat DAT in situ and the human DAT expressed by host cell systems such as C6 glioma and HEK 293 cells. Evidence for DAT dimers under non-reducing conditions was obtained in experiments in which photoaffinity-labeled rat DAT was immunoprecipitated with anti-DAT antibody. Cysteine mutants of the human DAT were prepared by site-directed mutagenesis. In the present application, we propose to further study the relationships between cocaine binding and the substrate recognition domains on the DAT including the role of cysteine residues. Human DAT cysteine mutants will be prepared and tested for DAT cell surface expression (biotinylation), DAT function (DA uptake and blocker binding), sensitivity towards sulfhydryl reagents (membrane permeable and impermeable), and their capability to form dimers (co-transfection of wild-type and mutant hDAT cDNA). The impact of palmitoylation of cysteine residues on DAT function will be studied, both in the human DAT expressed in C6 glioma cells and the in situ rat DAT. The interaction of Na+, K+, C1- and H+ with hDAT function will be assessed in preparations of membranes or plasma membrane vesicles of cells expressing the human DAT. The latter system will allow studying the impact of ion gradients and membrane potential on DAT function as well as determining which form of DA, neutral or protonated, is translocated. Finally, the spare receptor concept will be tested for the DAT by classical pharmacological approaches involving irreversible blockade by the cocaine analog RTI-76 as well as molecular techniques addressing oligomerization, both for the human DAT in a host system and the rat DAT in situ.